Promoter analysis

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Promoter analysis

• Bonehead Seminar
• Anne Seppänen
• 8.10.03

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TF transcription factor

• binding sites 5-25bp
• proteins
• two or more repeated binding sites
• combinatorial clusters
• TF-database, TRANSFAC

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TRANSFAC

• database of cis-acting DNA elements and
  trans-acting factors
• collection of in vitro results
• printed compilation 1988,
• computer-readable format 1990
• PWM Position Weight Matrix

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Regulatory regions, where are they?

• 5 upstream
• 100 - 100 000bp away from 1st exon
• 3 downstream
• intron regions (especially 1st intron)
• How long are they?
• Evolutionarily conserved

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(No Transcript)
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5 osteoclast specific genes

• TRACP
• RANK
• CA2
• Calcitonin receptor
• Cathepsin K

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Promoter analysis

• different algorithms and tools for searching
  TF-binding sites
• - many false negative and positive results
• use of only PWM a poor resource
• How to approve?
• - search conserved non-coding regions
• - use of expression data

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Vista analysis tool

• mVista
• - global alignment for given sequences
• - conserved non-coding regions
• rVista
• - search for TF-binding sites from TRANSFAC
  database

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Example of rVista result file
1 VTBP_01 - 1-8 gtTTTATa 0.923 1.000 610-617
gtTTTATa 0.923 1.000 90.00 2 VEN1_01 1-7
GTTTTat 0.893 0.922 610-616 GTTTTat 0.893 0.922
90.00 3 VCDXA_01 2-8 TTTTAta 0.956 0.968
611-617 TTTTAta 0.956 0.968 90.00 4 VCDXA_02
2-8 tTTTATa 0.963 1.000 611-617 tTTTATa 0.963
1.000 90.00 5 VUSF_02 - 1529-1542 cctacACGTGacag
0.971 1.000 2911-2924 gcttcAGGTGgcag 0.927 0.935
80.00 6 VUSF_02 1529-1542 cctaCACGTgacag 0.971
1.000 2911-2924 gcttCAGGTggcag 0.927 0.935
80.00 7 VUSF_Q6 - 1531-1540 tacaCGTGAc 0.955
1.000 2913-2922 ttcaGGTGGc 0.887 0.913 80.00 8
VCAP_01 - 1532-1539 acaCGTGA 0.942 0.946
2914-2921 tcaGGTGG 0.964 0.971 80.00 9 VUSF_C -
1532-1539 acACGTGa 0.995 1.000 2914-2921 tcAGGTGg
0.935 0.918 80.00 10 VCAP_01 1532-1539
ACACGtga 0.928 0.935 2914-2921 TCAGGtgg 0.944
0.950 80.00 11 VGFI1_01 - 1533-1556
cacgtgacaggTGATTtatctatg 0.909 1.000 2915-2938
caggtggcaggTGATTaatgtgca 0.867 1.000 80.00 12
VUSF2_Q6 1533-1538 CACGTg 1.000 1.000
2915-2920 CAGGTg 0.989 0.986 80.00 13 VE47_02
1535-1550 cgtgaCAGGTgattta 0.940 1.000 2917-2932
ggtggCAGGTgattaa 0.934 1.000 80.00 14 VUSF_02 -
1536-1549 gtgacAGGTGattt 0.948 0.935 2918-2931
gtggcAGGTGatta 0.947 0.935 80.00 15 VUSF_02
1536-1549 gtgaCAGGTgattt 0.948 0.935 2918-2931
gtggCAGGTgatta 0.947 0.935 80.00 16 VAREB6_01 -
1537-1549 tgaCAGGTgattt 0.925 1.000 2919-2931
tggCAGGTgatta 0.912 1.000 80.00 17 VAREB6_02 -
1537-1548 tgacAGGTGatt 0.994 1.000 2919-2930
tggcAGGTGatt 0.979 1.000 80.00 18 VAREB6_03 -
1537-1548 tgaCAGGTgatt 0.929 1.000 2919-2930
tggCAGGTgatt 0.906 1.000 80.00 19 VP53_02 -
1537-1546 tgACAGGtga 0.875 0.965 2919-2928
tgGCAGGtga 0.875 0.965 80.00 20 VLMO2COM_01
1537-1548 tgaCAGGTgatt 0.956 1.000 2919-2930
tggCAGGTgatt 0.958 1.000 80.00 21 VMYOD_01
1537-1548 tgaCAGGTgatt 0.947 1.000 2919-2930
tggCAGGTgatt 0.926 1.000 80.00 22 VDELTAEF1_01 -
1538-1548 gacAGGTGatt 0.979 1.000 2920-2930
ggcAGGTGatt 0.979 1.000 80.00 23 VMYOD_Q6 -
1538-1547 gacAGGTGat 0.995 1.000 2920-2929
ggcAGGTGat 0.992 1.000 80.00 24 VSREBP1_01 -
1538-1548 gacAGGTGatt 0.914 0.937 2920-2930
ggcAGGTGatt 0.916 0.937 80.00 25 VUSF_Q6 -
1538-1547 gacaGGTGAt 0.913 0.926 2920-2929
ggcaGGTGAt 0.909 0.926 80.00 26 VE12_Q6
1538-1548 gaCAGGTgatt 0.959 1.000 2920-2930
ggCAGGTgatt 0.969 1.000 80.00 27 VMYOGENIN_Q6
1538-1545 gaCAGGTg 0.982 1.000 2920-2927 ggCAGGTg
1.000 1.000 80.00 28 VCAP_01 - 1539-1546
acaGGTGA 0.978 0.982 2921-2928 gcaGGTGA 0.977
0.982 80.00 29 VUSF_C - 1539-1546 acAGGTGa 0.932
0.918 2921-2928 gcAGGTGa 0.934 0.918 80.00 30
VCAP_01 1539-1546 ACAGGtga 0.933 0.939
2921-2928 GCAGGtga 0.935 0.942 80.00 31 VPAX4_03
- 1540-1551 cAGGTGatttat 0.876 0.903 2922-2933
cAGGTGattaat 0.878 0.903 80.00
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Some results of

• 4 osteoclast specific genes
• 5000bp 5 upstream sequences
• human and mouse comparison conserved non-coding
  regions
• clustering those TFs that have binding site on
  conserved regions

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Alternative splicing sites for Exon1 of TRACP
(Walsh N et al. 2003 Gene)
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Conserved non-coding regions
ca2
cathepsin K
calcitonin receptor
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The most usual TF

• cathepsin K, calcitonin receptor and ca2
• - CAP_01 and OCT_03
• - GEN_INI_B and STAT
• rank
• - GATA

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Clustering of TFBSs
Defaults min 2 TFBSs no
overlaping max 5bp distance
between two TFs
When two clusters are similar?
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Ca2
GFI1
GEN CAP
AP3 STAT
EN1 CAP
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Cathepsin K
STAT (CDXA)
OSF2 CDXA
CAP PAX4
CAP PAX4
CAP PAX2
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Calcitonin receptor
HNF3A HNF3B FOXD3 HFH3 CIZ PAX2
CDXA PAX2 TBP
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Rank
OCT1 NKX62 PAX2 MEF2 CDXA HNF3A
CDXA PAX2 (NKX62/CRX) (OCT1)
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Future plan

• Learn more about
• -biology and genes in general
• -other databases and tools
• -promoter analysis
• TRACP needs special handling in
• promoter analysis